The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3′ non-coding region of the genome

AbstractTick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E315) and NS5 (NS5654,655) proteins, and into the 3′ non-coding region (Δ30) of TBEV/DEN4. The variant that contained all three mutations (vΔ30/E315/NS5654,655) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vΔ30/E315/NS5654,655 should be further evaluated as a TBEV vaccine

Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection

The molecular immunopathogenesis of West Nile virus (WNV) infection is poorly understood. Here, we characterize a mouse model for WNV using a subcutaneous route of infection and delineate leukocyte subsets and immunoregulatory factors present in the brains of infected mice. Central nervous system (CNS) expression of the chemokine receptor CCR5 and its ligand CCL5 was prominently up-regulated by WNV, and this was associated with CNS infiltration of CD4+ and CD8+ T cells, NK1.1+ cells and macrophages expressing the receptor. The significance of CCR5 in pathogenesis was established by mortality studies in which infection of CCR5−/− mice was rapidly and uniformly fatal. In the brain, WNV-infected CCR5−/− mice had increased viral burden but markedly reduced NK1.1+ cells, macrophages, and CD4+ and CD8+ T cells compared with WNV-infected CCR5+/+ mice. Adoptive transfer of splenocytes from WNV-infected CCR5+/+ mice into infected CCR5−/− mice increased leukocyte accumulation in the CNS compared with transfer of splenocytes from infected CCR5−/− mice into infected CCR5−/− mice, and increased survival to 60%, the same as in infected CCR5+/+ control mice. We conclude that CCR5 is a critical antiviral and survival determinant in WNV infection of mice that acts by regulating trafficking of leukocytes to the infected brain

West Nile Virus Spreads Transsynaptically within the Pathways of Motor Control: Anatomical and Ultrastructural Mapping of Neuronal Virus Infection in the Primate Central Nervous System

<div><p>Background</p><p>During recent West Nile virus (WNV) outbreaks in the US, half of the reported cases were classified as neuroinvasive disease. WNV neuroinvasion is proposed to follow two major routes: hematogenous and/or axonal transport along the peripheral nerves. How virus spreads once within the central nervous system (CNS) remains unknown.</p><p>Methodology/Principal Findings</p><p>Using immunohistochemistry, we examined the expression of viral antigens in the CNS of rhesus monkeys that were intrathalamically inoculated with a wild-type WNV. The localization of WNV within the CNS was mapped to specific neuronal groups and anatomical structures. The neurological functions related to structures containing WNV-labeled neurons were reviewed and summarized. Intraneuronal localization of WNV was investigated by electron microscopy. The known anatomical connectivity of WNV-labeled neurons was used to reconstruct the directionality of WNV spread within the CNS using a connectogram design. Anatomical mapping revealed that all structures identified as containing WNV-labeled neurons belonged to the pathways of motor control. Ultrastructurally, virions were found predominantly within vesicular structures (including autophagosomes) in close vicinity to the axodendritic synapses, either at pre- or post-synaptic positions (axonal terminals and dendritic spines, respectively), strongly indicating transsynaptic spread of the virus between connected neurons. Neuronal connectivity-based reconstruction of the directionality of transsynaptic virus spread suggests that, within the CNS, WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons.</p><p>Conclusions/Significance</p><p>This study offers a new insight into the neuropathogenesis of WNV infection in a primate model that closely mimics WNV encephalomyelitis in humans. We show that within the primate CNS, WNV primarily infects the anatomical structures and pathways responsible for the control of movement. Our findings also suggest that WNV most likely propagates within the CNS transsynaptically, by both, anterograde and retrograde axonal transport.</p></div

Summary of neurological functions related to the structures containing WNV-labeled neurons.

<p>Summary of neurological functions related to the structures containing WNV-labeled neurons.</p

WNV-labeled neurons in the spinal cord.

<p>Representative images of WNV-labeled neurons in the cervical (<b>A</b> and <b>D</b>), thoracic (<b>B</b> and <b>E</b>), and lumbar regions (<b>C</b> and <b>F</b>) of the spinal cord are shown on indicated dpi. Approximate boundaries of the spinal cord gray matter are outlined in the overview images. Clarke’s column is shown by magenta overlay in <b>E</b>. Round insets show the corresponding circled areas at higher magnification. Note that the majority of WNV-labeled neurons occupy a medial portion of the Clarke’s column. Vh, ventral horns; CC, Clarke’s column. Bars in overview images: 1000 μm. Bars in the round insets: 100 μm.</p

WNV particles versus dense core vesicles in the spinal cord.

<p>Shown are electron microscopy images of ventral horns of cervical and lumbar regions of the spinal cord. (<b>A</b>) Four large vesicles containing virions are shown by yellow overlays. These vacuoles lie close to a degenerating myelin (My) sheath of axon. Degenerating part of the myelin sheath is shown by magenta overlay. (<b>B</b>) Higher magnification image of the boxed area in (<b>A</b>) shows several virions of ~ 40 nm in diameter. Note a lighter lipid layer underneath the darker envelope layer especially clearly visible in one of the virions (arrow). (<b>C</b> and <b>D</b>) Compare two axonal areas containing virions (yellow overlay in <b>C</b>) or dense core vesicles (<b>D</b>). Insets show red circled areas at higher magnification. Approximate diameters of virions and vesicles are indicated. Scale bars: 100 nm (additional scale bars for dense core vesicles are indicated within the inset in <b>D</b>).</p